Phylogeny, Evolution of Lichenivory, and Chemical Sequestration in the Lichen Moth Genus Hypoprepia Hubner (Insecta: Lepidoptera: Erebidae: Arctiinae: Lithosiini)
Tiger moths (Lepidoptera: Erebidae: Arctiinae) are a diverse and ideal study system for understanding plant-insect interactions, and the evolution of antipredatory behaviors, because they are known to sequester plant toxins (secondary metabolites). Significant progress has been made in developing evolutionary hypotheses of anti-predatory defenses in derived tiger moth lineages, but critical knowledge gaps in our understanding of how these strategies may have evolved across all lineages still exist. For example, Lithosiini are a relatively unique clade of tiger moths that are known to feed on mosses, liverworts, lichens, and algae. Some of the lichens that are fed upon by lithosiines are known to produce toxic secondary metabolites. It has been demonstrated that lithosiines cope with the secondary metabolites, and that some secondary metabolites are also sequestered by the caterpillar for which their function is poorly understood. Data suggests adults of some lichen-feeding Lithosiini are unpalatable to vertebrate predators and may couple chemical protection with acoustic signaling. Thus, lichen-lichenivore interactions in this clade may provide important clues to the origins of defensive chemical sequestration within arctiines. Little is known about the phylogenetic relationships among the Lithosiini, which precludes our understanding of the evolution of other chemically-mediated behaviors in tiger moths. For example, the lichen moth genus Hypoprepia is widely distributed throughout North America and Mexico for which five species are described. The Hypoprepia have been the subject of studies that have demonstrated the presence of putative lichen chemicals in the adults, they have been documented to be unpalatable to bats as adults, and click in response to bat sonar which has been attributed to chemical defense as acoustic aposematism. Yet throughout the range of Hypoprepia, many adult color forms exist which has confounded accurate identification and called into question the current definitions for species limits within the genus. Thus, Hypoprepia can be viewed as both an emerging model to further test hypotheses on chemical relationships between insects and their hosts, specifically with regards to lichen-invertebrate interactions which are poorly understood, and also as a system limited by inaccurate species limits and improper phylogenetic resolution. A robust and accurate phylogeny will be unattainable if the genera under examination are not monophyletic.^ In this dissertation, I sampled species of the widely distributed North American lichen moth genus Hypoprepia Hübner in order to determine their phylogenetic placement among other lichen moth genera hypothesized to share common ancestry, clarify species limits within the genus, and examine their chemical relationship with their lichen hosts. Morphological characters were scored and analyzed using Maximum Parsimony to determine sister relationships within a phylogenetic complex for Hypoprepia and proposed related genera. I also sought to clarify species limits in phenotypically entangled and sympatric H. fucosa-minata complex. An integrative approach to species delineation was undertaken that utilizes morphological, molecular, chemical, distributional, and behavioral data for the Hypoprepia fucosa-minata complex. Next-generation sequencing (WideSeq) was employed to generate a robust phylogenetic hypothesis for this complex using Bayesian Inference and Maximum Likelihood analyses.^ In addition to the phylogenetic reconstructions, I utilized advanced microscopy (SEM) coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) in order to develop novel character systems for larvae to be used in comparative analyses for Hypoprepia. During these investigations, a novel structure, possibly used in chemical defense, was discovered on late instar Hypoprepia larvae. I tested the presumed defensive function of these structures by analyzing liquid exudates for the presence of lichen-derived metabolites. Results indicated that a putative lichen metabolite, methyl orsellinate, was highly concentrated in the exudate relative to the whole body of the caterpillars. These results suggest that Hypoprepia are utilizing lichen secondary metabolites in a manner previously unknown to science. No other animal is known to sequester and utilize lichen secondary metabolites in a secretion for a possibly defensive function. The implications of these results with regards to the evolution of chemical sequestration among the lithosiines are discussed.^ In order to investigate subtleties of chemical sequestration among life history stages within a lichen moth species, I employed a metabolomics approach to test the hypothesis that lichen-derived metabolites are unequally distributed among life stages and that lab-reared moths have less metabolite diversity than wild-caught individuals using the lichen moth Crambidia cephalica (Grote and Robinson). Results indicated that several putative lichen-derived metabolites were detected among the life stages of C. cephalica. Additionally, different lichen-derived metabolites were present and/or absent when compared among the different life stages and the lichen host. These results provide evidence that there are multiple lichen-derived metabolites sequestered by C. cephalica, some metabolites are retained through to adulthood and that others are lost after the larval and pupal stages. The presence of lichen-derived metabolites in discrete life stages may indicate functional properties of the metabolites for C. cephalica with regards to chemical protection from antagonists and other physiological processes.^
Jennifer M. Zaspel, Purdue University.
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